Cystic Fibrosis (CF) is an inherited disorder with major respiratory morbidity and mortality, for which there is no cure. Mutations in the CF gene lead to impaired chloride and water secretion across the airway epithelium. The goal for this proposal is the stable transduction of neonatal lungs with the normal CF gene, CFTR, using a recombinant parvovirus, adeno-associated virus, AAV. The HYPOTHESIS is that AAV gene therapy vectors will achieve higher level stable expression when instilled in the neonatal lung than after delivery to the adult animal. The underlying rationale is that the neonatal lung will have a greater number of proliferating cells because it is undergoing rapid growth, and thus will be more effectively transduced with the viral vectors. The first experiments will use the reporter gene construct, AAV-lacZ, whose expression can be demonstrated histochemically as a blue stain. Part I of this grant is directed towards achieving expression of the AAV gene therapy vectors in the lungs of neonatal animals. Aim 1 is to instill AAV- lacZ into the lungs of newborn (3-4 day) rabbits by direct tracheal puncture, and then to study lacZ expression in the pulmonary epithelium. Aim 2 is to instill AAV-lacZ into the amniotic sacs of day 28-30 fetal rabbits, and determine at birth the cell types expressing lacZ. Proliferating cells will be identified by in vivo nuclear labeling with bromo-deoxyuridine (BRDU). Quantitation of the proliferation index and of lacZ expression will be pursued using videoimaging software. Long term expression will be assessed with the AAV-CFTR vectors. Part II will use both AAV-CFTR and AAV-lacZ and study the direct consequences of the vector to airway and alveolar development, indices of inflammation, and safety of the animal. Aim 3 is to determine whether AAV viral vectors induce inflammation in the lung. Morphological analysis at both the light and electron microscopic levels, bronchoalveolar lavage, and assessment of interleukin (Il-6) expression will be accomplished. Issues to be addressed include efficiency, stability in the pulmonary epithelium, and safety. The five year objective of this grant is to establish a gene therapy protocol that could eventually be tested in the CF mouse model, primates, and applied to the human infant with CF.